SUMMARY The breast cancer type I susceptibility protein (BRCA1) and BRCA1-associated RING domain protein I (BARD1) heterodimer promote genome integrity through pleiotropic functions, including DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1-BARD1 heterodimerization is required for their mutual stability, HR function, and role in tumor suppression; however, the upstream signaling events governing BRCA1-BARD1 heterodimerization are unclear. Here, we show that SIRT2, a sirtuin deacetylase and breast tumor suppressor, promotes BRCA1-BARD1 heterodimerization through deacetylation. SIRT2 complexes with BRCA1-BARD1 and deacetylates conserved lysines in the BARD1 RING domain, interfacing BRCA1, which promotes BRCA1-BARD1 heterodimerization and consequently BRCA1-BARD1 stability, nuclear retention, and localization to DNA damage sites, thus contributing to efficient HR. Our findings define a mechanism for regulation of BRCA1-BARD1 heterodimerization through SIRT2 deacetylation, elucidating a critical upstream signaling event directing BRCA1-BARD1 heterodimerization, which facilitates HR and tumor suppression, and delineating a role for SIRT2 in directing DSB repair by HR.

The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response–dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11 , and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress–induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.

Background: Human Rad51 (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA) in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPγS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding.

Human Rad52 (hRad52) and Rad51 (hRad51) proteins are important components of homologous recombination machinery involved in DNA double strand break repair. hRad52 subunits oligomerize to form rings, which are further believed to stack one over another giving rise to higher order structures. Such structures bind the ends of duplex DNA to bring about DNA end joining. hRad51 exists in the native state as oligomeric rings and monomerizes to interact with the DNA. In our current study, we report disruption and solubilization of hRad52 aggregates and higher order aggregation of hRad51 molecules at high salt (KCl) concentration. Computational analysis of the crystal structure available for N-terminal 212 amino acids of hRad52 protein reveal a dense unique distribution of salt bridges, not only between adjacent but also between penultimate subunit neighbors which perhaps contribute to stabilization of hRad52 oligomeric rings. Our results suggest that disruption of inter-subunit salt bridges and thereby perturbation of interaction between individual monomers as the underlying mechanism for salt mediated monomerization of hRad52 protein. The crystal structure of Rad51 on the other hand lacks such dense salt-bridge connectivity suggesting that salt-mediated monomerization is a feature of proteins with dense salt-bridge networks. Salt brings together the hydrophobic surface residues of hRad51 in a process termed as "salting out" resulting in aggregation of hRad51 molecules. Given the functional relevance of oligomeric hRad52 and monomeric hRad51 in homologous recombination mediated repair, our findings imply that salt regulates the oligomerization status of these repair proteins, and thereby, their functions respectively.

The breast cancer type I susceptibility protein (BRCA1) and BRCA1-associated RING domain protein I (BARD1) heterodimer promotes genome integrity through pleiotropic functions, including DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1-BARD1 heterodimerization is required for their mutual stability, HR function, and role in tumor suppression; however, the upstream signaling events governing BRCA1-BARD1 heterodimerization are unclear. Here, we show that SIRT2, a sirtuin deacetylase and breast tumor suppressor, promotes BRCA1-BARD1 heterodimerization through deacetylation. SIRT2 complexes with BRCA1-BARD1 and deacetylates conserved lysines in the BARD1 RING domain, interfacing BRCA1, which promotes BRCA1-BARD1 heterodimerization and consequently BRCA1-BARD1 stability, nuclear retention, and localization to DNA damage sites, as well as efficient HR. Our findings define a mechanism for regulation of BRCA1-BARD1 heterodimerization through SIRT2 deacetylation, elucidating a critical upstream signaling event directing BRCA1-BARD1 heterodimerization, which facilitates HR and tumor suppression, and delineating a role for SIRT2 in directing DSB repair by HR. Citation Format: Elizabeth V. Minten, Priya Kappor-Vazirani, Chunyang Li, Hui Zhang, Kamakshi Balakrishnan, David S. Yu. SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-002.